Radical SAM catalysis via an organometallic intermediate with an Fe–[5′-C]-deoxyadenosyl bond by Masaki Horitani, Krista Shisler, William E. Broderick, Rachel U. Hutcheson, Kaitlin S. Duschene, Amy R. Marts, Brian M. Hoffman, and Joan B. Broderick Science Volume 352(6287):822-825 May 13, 2016 Published by AAAS

Fig. 1 Activation of PFL by PFL-AE, with concomitant cleavage of SAM to methionine and 5′-deoxyadenosine. Activation of PFL by PFL-AE, with concomitant cleavage of SAM to methionine and 5′-deoxyadenosine. Masaki Horitani et al. Science 2016;352:822-825 Published by AAAS

Fig. 2 EPR spectra showing the formation of the PFL glycyl radical (G•) from Ω. EPR spectra showing the formation of the PFL glycyl radical (G•) from Ω. (A) (Top) EPR spectra of mixture of photoreduced PFL-AE and PFL/SAM freeze-quenched at ~77 K (500 ms), stored at 77 K, and then annealed at progressively higher temperatures for the indicated times (see supplementary materials). At 12 K, the G• radical spectrum is highly saturated and its amplitude diminished; at 40 K, the signal from the rapidly relaxing Ω is correspondingly diminished. The spectra here have had the residual intensities at both temperatures subtracted out (fig. S2), with one exception: Ω is completely lost after annealing at 220 K; the dashed curve shows the residual signal from the saturated G• radical. (Bottom) Populations of Ω and the G• radical, relative to the final (220 K) G• radical concentration taken as 100% (Sum), as derived from EPR spectra (see supplementary materials). (B) X-band EPR spectra for photoreduced PFL-AE freeze-quenched (77 K) 500 ms after mixing with PFL G734A/SAM, with spectra collected at 12 and 40 K. Conditions: microwave frequency = 9.23 GHz, microwave power = 1 mW, 100-kHz modulation amplitude = 8 G, and T, as indicated. The gain at a given T is fixed. Masaki Horitani et al. Science 2016;352:822-825 Published by AAAS

Fig. 3 35-GHz ENDOR spectra at g⊥ for photoreduced PFL-AE freeze-quenched with PFL/SAM. 35-GHz ENDOR spectra at g⊥ for photoreduced PFL-AE freeze-quenched with PFL/SAM. To first order, an ENDOR spectrum of a spin I = 1/2 nucleus (N) in a frozen solution comprises a superposition of signals from different orientations, each signal a doublet at frequencies ν± = |ν(N) ± A/2|, where ν(N) is the nuclear larmor frequency and A is the orientation-dependent hyperfine coupling (23). For 13C, A/2 << ν(13C), and it is convenient to plot spectra versus ν – ν(13C). For 57Fe, ν(57Fe) << A/2, and spectra are plotted versus ν. (A) 13C CW ENDOR for [adenosyl-13C10] SAM. The green dashed lines denotes the best-match simulation to the axial hyperfine tensor (see supplementary materials). Simulation parameters: aiso = 9.4 MHz, 2T = 5.3 MHz, and β = 90°. Conditions: microwave frequency = 35.39 GHz, microwave power = 1 mW, 100-kHz modulation amplitude = 1.3 G, rf sweep rate = 1 MHz/s, and T = 2 K. (Inset) Mims ENDOR spectrum. Conditions: microwave frequency = 35.20 GHz; MW pulse length, (π/2) = 50 ns; τ = 500 ns; and T = 2 K. (B) Mims ENDOR spectrum from [methyl-13C] SAM. Conditions: microwave frequency = 35.08 GHz; MW pulse length, (π/2) = 50 ns; τ = 500 ns; and T = 2 K. (C) 57Fe CW ENDOR for 57Fe-enriched Ω and photoreduced PFL-AE. (Top) CW ENDOR spectra for 57Fe-enriched (red) and natural-abundance (gray) rfq samples. (Bottom) Frequency sweep and randomly hopped stochastic CW ENDOR spectra (23) for 57Fe-enriched reduced PFL-AE. Conditions: microwave frequency = 35.45 GHz and 35.07 GHz for rfq and 57Fe-enriched reduced PFL-AE, respectively; microwave power = 1 mW; 100-kHz modulation amplitude = 1.3 G; rf sweep rate = 1 MHz/s; stochastic CW ENDOR cycle, rf-on = 3 ms, rf-off = 1 ms; sample collection time = 3 ms; and T = 2 K. See supplementary materials for details. Masaki Horitani et al. Science 2016;352:822-825 Published by AAAS

Fig. 4 Model for bio-organometallic intermediate Ω. Model for bio-organometallic intermediate Ω. Whether methionine remains coordinated to the unique iron site is not currently known. Blue, nitrogen; white, carbon; red, oxygen; yellow, sulfur; orange, iron. Masaki Horitani et al. Science 2016;352:822-825 Published by AAAS